Effect of LSD on mitotic and meiotic plant chromosomes

LSD was found to induce chromosomal aberrations in root tip cells of Allium cepa, Hordeum vulgare and Secale cereale. Aberrations occurred in the form of chromatid and isochromatid breaks with most of these breaks failing to rejoin. The distribution of chromosome breaks was not uniform over the length of chromosomes, and a majority of the breaks were localized at the centromeric regions. For a given dose of LSD (30 g/ml), onion appeared to be more susceptible than barley or rye. The diploid and tetraploid rye used in the study showed no appreciable difference in sensitivity to LSD treatment. — A preliminary study on meiotic chromosomes in LSD-treated diploid rye revealed the presence of univalents, chromosome breaks and fragments, suggesting that LSD can induce meiotic abnormalities in plant material.Contribution from the Department of Agronomy, University of Kentucky. The investigation reported in this paper (73-3-75) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director.     

Grades of chromatid organisation in mitotic and meiotic chromosomes

It is suggested that not one, but four different grades of lampbrush chromosome organisation characterise different stages of mitosis and meiosis: (a) where a single chromatid organises symmetrically disposed lateral loops (second meiotic prophase), (b) where the two sister chromatids of a visibly single chromosome organise lateral loops in a laterally asymmetrical fashion, both sets of loops projecting from the same side and away from the face used, in meiosis, for pairing (early first meiotic prophase), (c) where the lateral loops organised by two visibly separate sister chromatids are symmetrically disposed on either side of the chromosome and project away from each other (mitotic prophase and late first meiotic prophase), (d) where the organisation is as in (c) but chromatid axes are intimately fused and form a visibly single strand (female amphibian diffuse diplotene).     

Grades of chromatid organisation in mitotic and meiotic chromosomes

The different grades of chromatid organisation recognised in mitotic and meiotic chromosomes are interpreted in terms of a new master/slave model, in which a linear strand of master loci has an associated slave assembly of chromomeres. These are joined by labile interchromomeric linkages, which might be short overlap segments of single-stranded DNA. Each slave assembly, or chromomere, is considered to give rise to a lateral slave loop. The model is able to explain how transitions between the four basic types of lampbrush organisation could occur at mitosis and meiosis, how the presence or absence of transitions at early first meiotic prophase may decide whether or not synapsis may take place, and why it should be restricted to two homologues at any one point. It provides a basis for understanding how precision hybrid DNA cross-over events involving single DNA molecules could be achieved in a regular, repeatable and controlled fashion, between such relatively massive structures as chromosomes, and how resolution of the primary exchange events could lead to the development of chiasmata. It also suggests a new way of thinking about the mechanisms underlying the separation and rotation of chromosome arms which occurs during the diplotene and diakinesis stages of most chiasmate species. Semi-conservative DNA replication, iso-labelling and breakage and reunion studies which suggest only one axial strand are also explicable in terms of the model, as is the occurrence of side-arm bridges, involving two visible “sub-chromatid” strands, at anaphase stages.     

Differential associative behaviour of mitotic and meiotic acrocentric chromosomes

Summary A comparative study of the association of mitotic acrocentric chromosomes and acrocentric bivalents at the pachytene stage shows that at least two factors can act in the associative behaviour of these chromosomes: (1) Nor activity and (2) the presence of satellite DNA in the short arms of these chromosomes. These factors do not act with the same intensity in the two cell lines studied. In lymphocytes, Nor activity prevails, whereas satellite DNA plays the main role in the association of acrocentric chromosomes in germ cells at the pachytene stage.     

The mitotic, polytene, and meiotic chromosomes of Drosophila ananassae.

The mitotic chromosome complement of D. ananassae consists of four structurally distinguishable submetacentric pairs and all four have been identified with their linkage groups. For the polytene chromosome complement of six arms representing the X, second and third chromosomes, an improved reference map has been constructed and used to describe selected cytogenetically useful rearrangements. In meiotic prophase of spermatocytes, chromosomes 2 and 3 form pachytene-diplotene bivalents whose arms may be associated by chiasmata in postdiplotene stages, but the X, Y and fourth chromosomes participate in a complex multivalent. No correlation was detected between meiotic chromosome behavior and specific genes that regulate crossing over in males. In male inversion heterozygotes having high levels of genetically monitored crossing over, no unequivocal evidence was found for formation of either pachytene inversion loops or anaphase bridges and fragments.     

Uptake of isolated plant chromosomes by plant protoplasts

For mass isolation of plant metaphase chromosomes, cultured cells of wheat (Triticum monococcum) and parsley (Petroselinum hortense) were synchronized by hydroxyurea and colchicine treatment. This synchronization procedure resulted in high mitotic synchrony, especially in suspension cultures of parsley in which 80% of the cells were found to be at the metaphase stage. Mitotic protoplasts isolated from these synchronized cell cultures served as a source for isolation of chromosomes. The described isolation and purification method yielded relatively pure chromosome suspension. The uptake of the isolated plant chromosomes into recipient wheat, parsley, and maize protoplasts was induced by polyethylene-glycol treatment. Cytological studies provided evidences for uptake of plant chromosomes into plant protoplasts.Abbreviations PEG polyethylene glycol - HU hydroxyruea - C colchicine - HUC hydroxyurea and colchicine - CIM chromosome isolation medium - TCM Tris chromosome medium     

Genetic selection of meiotic and mitotic recombinant yeast artificial chromosomes.

We have developed a genetic screen for the isolation of larger or smaller recombinant yeast artificial chromosomes derived from overlapping YACs. Integration plasmids were used to modify the TRP1 and URA3 auxotrophic markers present respectively on the left and right vector arms of one of the parental YACs. Diploids containing the two parental YACs were studied through meiosis and mitosis. Tetrad analysis revealed the presence of meiotic recombinant YACs at a frequency comparable with what is expected for yeast DNA (about 3 kb/cM). More direct genetic selection of diploids on -TRP-LYS synthetic media in the presence of 5-fluoro-orotic acid (5-FOA), led to the isolation of mitotic recombinant YACs at a high frequency. Analysis of these yeast cells by pulsed-field gel electrophoresis, confirmed the loss of both parental artificial chromosomes, and the specific retention of a larger or smaller recombinant YAC.     

Flow cytometric analysis and sorting of plant chromosomes from Petunia hybrida protoplasts

A method to obtain a high metaphase index and thereafter a plant chromosome suspension is described for Petunia hybrida (2n = 14). Mesophyll protoplast cultures have been used, giving easily disrupted cell walls and a high percentage of dividing cells after 42 h. On 2.5 mM colchicine-treated cells, metaphase indexes reaching 10% were routinely obtained. The lysis medium in which the protoplast-derived cells were disrupted was a simplified culture medium. After chromosome release, samples were stained with Hoechst 33342 dye and analysed by flow cytofluorometry. The histogram of fluorescence intensities included three peaks of metaphase chromosomes and a duplication of this flow karyotype provoked by "monochromatid chromosome." This interpretation was established after flow sorting; micronuclei could also be observed and sorted. Of the 7 chromosomes, only the largest formed a distinct peak while the others were incompletely resolved, due to the similar DNA content of various chromosomes. Model distributions of Petunia hybrida chromosomes have been computed according to the relative chromosome length. The theoretical histograms indicated that low variability is indispensable for resolving distinctive chromosome peaks. The experimental flow karyotype was consistent with one of the models having CV of 2.5%.     

An efficient procedure for isolation of nuclei from plant protoplasts

Summary The present communication describes an easy, efficient and rapid method for isolation of nuclei from plant protoplasts. Release of nuclei is accomplished by disruption of protoplasts in an appropriate buffer containing a very low concentration (0.01%) of the detergent Triton X-100. The pH of the nuclei isolation buffer (5.3) played a critical role in the recovery of stable nuclei in large numbers. Supplementation of buffer (10 mM MES) with spermine (0.1 mM), dithiothreitol (2.5 mM), ethylenediaminetetraacetic acid (2.5 mM) and Nad and KCl (10 mM each) improved nuclear yield and quality. With the method developed it is possible to routinely recover 95% nuclei from the protoplasts within 30 minutes. The nuclear preparations are of high purity with little detectable cytoplasmic contamination and no clumping of the nuclei. The structural integrity of the nuclei has been assessed and confirmed by Nomarski differential interference contrast optics and ultrastructural observations.     

Effect of LSD-25 on mitotic and meiotic chromosomes of mice and monkeys

Summary Chromosomal studies were carried out in bone marrow and testes of mice treated with lysergic acid diethylamide (LSD-25) in acute and chronic experiments, in blood cultures of rhesus monkeys (Macaca mulatta) treated with LSD-25 in vitro, and in blood cultures and testicular preparations of rhesus macaques treated with LSD. No increase in chromosomal damage was observed in bone marrow or testes, but all blood cultures treated with LSD in vitro and some of the blood cultures from rhesus macaques treated in vitro showed a significant increase in chromosomal breaks and rearrangements.Publication No. 397 of the Oregon Regional Primate Research Center, supported in part by Grants No. F00163 and MHI12214 of the National Institutes of Health.     

A rapid, simple method for nuclei isolation from plant protoplasts

A rapid, simple method for nuclei isolation and purification from soybean (Glycine max L. Merr.) protoplasts is described. The isolated nuclei exhibited active amino acid incorporation and RNA synthesis, but DNA synthesis was not detectable. Analysis by CsCl density gradient centrifugation showed that DNA isolated from nuclei had a single band, while DNA isolated from protoplasts consisted of three bands comprised of nuclear DNA, mitochondrial DNA, and chloroplast DNA.     

Staining air dried protoplasts for study of plant chromosomes

The air drying technique used in mammalian cytology was applied to isolated plant protoplasts for study of chromosomes. For cultured celery cells, this technique resulted in good spreads of metaphase chromosomes with high resolution. Mitotic chromosomes of Brassica species are relatively small, poor stained by common stains, and difficult to spread by the squash technique. In this study, however, the chromosomes of B. carinata in callus culture were spread well and stained clearly with Giemsa staining solution. The chromosome preparations by the present techniques should also be amenable to chromosome banding studies in plants.     

The plant-specific family of DNA-binding proteins containing three HMG-box domains interacts with mitotic and meiotic chromosomes

? The high mobility group (HMG)-box represents a DNA-binding domain that is found in various eukaryotic DNA-interacting proteins. Proteins that contain three copies of the HMG-box domain, termed 3 × HMG-box proteins, appear to be specific to plants. The Arabidopsis genome encodes two 3 × HMG-box proteins that were studied here. ? DNA interactions were examined using electrophoretic mobility shift assays, whereas expression, subcellular localization and chromosome association were mainly analysed by different types of fluorescence microscopy. ? The 3 × HMG-box proteins bind structure specifically to DNA, display DNA bending activity and, in addition to the three HMG-box domains, the basic N-terminal domain contributes to DNA binding. The expression of the two Arabidopsis genes encoding 3 × HMG-box proteins is linked to cell proliferation. In synchronized cells, expression is cell cycle dependent and peaks in cells undergoing mitosis. 3 × HMG-box proteins are excluded from the nuclei of interphase cells and localize to the cytosol, but, during mitosis, they associate with condensed chromosomes. The 3 × HMG-box2 protein generally associates with mitotic chromosomes, while 3 × HMG-box1 is detected specifically at 45S rDNA loci. ? In addition to mitotic chromosomes the 3 × HMG-box proteins associate with meiotic chromosomes, suggesting that they are involved in a general process of chromosome function related to cell division, such as chromosome condensation and/or segregation.     

Improved methods for the isolation of individual and clustered mitotic chromosomes

We have optimized procedures for the isolation of mitotic chromosomes from tissue culture cells. The first procedure is a rapid method for obtaining individual, structurally intact chromosomes suitable for analysis by electron microscopy. Further purification of these on Percoll gradients results in chromosomes free of cytoplasmic contamination, allowing biochemical characterization of the structural proteins and enzymatic activities intrinsic to mitotic chromosomes. A third procedure permits efficient, large-scale purification of chromosomes clustered together, referred to as a chromosomal cluster. The use of EDTA-containing polyamine buffers minimizes modifications of proteins and DNA during isolation and maintains the integrity of the chromosomal structure. The conditions which lead to the isolation of chromosomal clusters, as opposed to individual chromosomes, have been analyzed. Comparison of the gei patterns of proteins derived from individual chromosomes, as compared to clusters, identifies additional proteins in the latter pattern. These proteins could be involved in maintaining interchromosomal organization or positioning in the metaphase cell.     

The meiotic chromosomes of man

Summary Information was obtained on the chromosome number, and the behavior of autosomes as well as of the sex chromosomes in meiosis in human male germ cells derived from 25 Japanese patients, 4 to 79 years in age, who were hospitalized mostly due to epididymitis, prostate cancer, undescended testes or infertility.In 16 out of the 25 specimens, the chromosome numbers, 46 in 2n and 23 in n, were consistently established together with an XY sex-determining mechanism based on spermatogonial and spermatocyte divisions. No reliable counts were obtained from the remaining 9 cases, because of that they provided no cells for precise investigation.The X and Y chromosomes during the leptotene stage were observed as two separate heteropycnotic bodies lying along the inner wall of the nucleus, while at pachytene they formed a sex-vesicle after homologous pairing. At the diplotene, diakinesis and first metaphase the X and the Y appeared as an isopycnotic bivalent showing an end-to-end association, though there were some cells in which they remained as two separate entities free from contact. Evidence was presented that the X and the Y seemed to associate with each other at the distal end of the short arm of each element.One or sometimes two smallest autosomal bivalents tended to show rather precociously a chiasma-terminalization at the first metaphase.The metaphase chromosomes of the second spermatocytes were evident by the haploid number as well as by their widely diverged chromatids with a characteristic spiral configuration.The testicular materials under study contained in most cases polyploid cells with a considerable frequency in spermatogonia as well as in first and second spermatocytes. Giant sperm heads were observed not infrequently, mostly being abnormal in shape. No significant correlation was obtained between the frequency of polyploid cells and the age of patients so far studied.Contribution No. 679 from the Zoological Institute, Faculty of Science, Hokkaido University, Sapporo. — It is our pleasure to dedicate this paper to Professor Dr. Hans Bauer, Max-Planck-Institut für Meeresbiologie, Tübingen, in honor of his sixtieth birthday.     

Structural differentiation of the meiotic and mitotic chromosomes of the salamander,Ambystoma macrodactylum

The lampbrush chromosomes of the long-toed salamander, Ambystoma macrodactylum Baird, have been analysed and a map of the oocyte genome prepared. The location of C-bands and cold-induced-constrictions has been established in mitotic chromosomes and compared with the location of marker structures and chiasmata in several lampbrush bivalents. In the lampbrush chromosomes, C-bands are tentatively correlated with sphere-organizing loci and with regions of low chiasma frequency; cold-induced-constrictions are tentatively correlated with regions of high chiasma frequency. In general, in this salamander, C-bands do not coincide in position with cold-induced-constrictions. We have compared our results with those obtained by Callan (1966) in his investigation of chromosomes of the axolotl, Ambystoma mexicanum, and we present an analysis of the similarities and differences that are visible in the chromosome sets of these two ambystomatid species.